Hyper telomere recombination accelerates replicative senescence and may promote premature aging R. Tanner Hagelstroma,b, Krastan B. Blagoevc,d, Laura J. Niedernhofere, Edwin H. Goodwinf, and Susan M. Baileya,1 aDepartment of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523-1618; bPharmaceutical Genomics Division, Translational Genomics Research Institute, Phoenix, AZ 85004; cNational Science Foundation, Arlington, VA 22230; dDepartment of Physics Cavendish Laboratory, Cambridge University, Cambridge CB3 0HE, United Kingdom; eDepartment of Microbiology and Molecular Genetics, University of Pittsburgh, School of Medicine and Cancer Institute, Pittsburgh, PA 15261; and fKromaTiD Inc., Fort Collins, CO 80524 Edited* by José N. Onuchic, University of California San Diego, La Jolla, CA, and approved August 3, 2010 (received for review May 7, 2010) Werner syndrome and Bloom syndrome result from defects in the cell-cycle arrest known as senescence (12). Most human tissues lack RecQ helicases Werner (WRN) and Bloom (BLM), respectively, and sufficient telomerase activity to maintain telomere length through- display premature aging phenotypes. Similarly, XFE progeroid out life, limiting cell division potential. The majority of cancers syndrome results from defects in the ERCC1-XPF DNA repair endo- circumvent this tumor-suppressor mechanism by reactivating telo- nuclease. To gain insight into the origin of cellular senescence and merase (13), thus removing telomere shortening as a barrier to human aging, we analyzed the dependence of sister chromatid continuous proliferation. In some situations, a recombination- exchange (SCE) frequencies on location [i.e., genomic (G-SCE) vs. telo- based mechanism known as “alternative lengthening of telomeres” meric (T-SCE) DNA] in primary human fibroblasts deficient in WRN, (ALT) maintains telomere length in the absence of telomerase (14). BLM, or ERCC1-XPF. Consistent with our other studies, we found evi- Chromosome orientation (CO)-FISH (15) is a strand-specific dence of elevated T-SCE in telomerase-negative but not telomerase- modification of standard FISH capable of providing information positive backgrounds. In telomerase-negative WRN-deficient cells, not obtainable by any other means, including detection of sister T-SCE—but not G-SCE—frequencies were significantly increased com- chromatid exchange (SCE) recombination between telomeres, pared with controls. In contrast, SCE frequencies were significantly events termed T-SCE (16). T-SCE are emerging as important elevated in BLM-deficient cells irrespective of genome location. In features of certain telomerase-negative backgrounds, such as ERCC1-XPF-deficient cells, neither T- nor G-SCE frequencies differed ALT (17) and early embryogenesis before activation of telomer- from controls. A theoretical model was developed that allowed an ase (18). In the context of combined Werner (WRN) helicase and in silico investigation into the cellular consequences of increased telomerase deficiency in the mouse, both central features of hu- T-SCE frequency. The model predicts that in cells with increased man Werner syndrome (WS) pathogenesis, elevated T-SCE was T-SCE, the onset of replicative senescence is dramatically accelerated observed, with higher rates associated with greater immortaliza- “ even though the average rate of telomere loss has not changed. Pre- tion potential (19). Conditional deletion of the protection of ” mature cellular senescence may act as a powerful tumor-suppressor telomeres (Pot)1a single-stranded telomere-binding protein has mechanism in telomerase-deficient cells with mutations that cause been shown to elicit a DNA damage response at mouse telo- T-SCE levels to rise. Furthermore, T-SCE-driven premature cellular se- meres, as well as aberrant homologous recombination manifested nescencemaybeafactorcontributing to accelerated aging in Werner as increased T-SCE (20). Human POT1 stimulates the RecQ and Bloom syndromes, but not XFE progeroid syndrome. helicases WRN and Bloom (BLM) to unwind telomeric DNA (21). Furthermore, in the absence of WRN, leading- and lagging- T-SCE | WRN | BLM | ERCC1-XPF | Monte Carlo strand DNA synthesis are uncoupled at replication forks, and POT1 is required for efficient replication of C-rich telomere strands (22). Such studies imply a relationship between T-SCE nderstanding the molecular processes that drive cellular rate and proliferative potential. However, the complexity of the Ureplicative senescence is critical to understanding human process—which entails a large number of telomeres, an ongoing longevity. Much attention has focused on telomere length as exchange process throughout colony growth, exponentially in- a determinant of replicative capacity (1). Telomeres are nucleo- creasing cell number, and independent assortment of chromatids fi protein structures composed of species-speci c tandemly re- into daughter cells during cell division—precludes a simple ana- peated, G-rich DNA sequences that cap and protect chromosome lytical approach to understanding this relationship. We therefore ends from nucleolytic attack and degradation (2). Telomeres also turned to theoretical methods in the search for a causal connec- conceal linear chromosome ends from inappropriate attempts at tion between T-SCE and cell proliferation. double-strand break (DSB) repair that might otherwise join Homologous recombination provides the most plausible chromosomes end-to-end. The preservation of natural chromo- mechanism by which telomeric DNA could be exchanged between some ends and the rejoining of broken DNA ends, both of which sister telomeres, and in the case of T-SCE, homology can be found are essential for preserving genomic integrity, rely on a common at many points on the sister chromatid. Thus, T-SCE need not subset of proteins (3), and both decline with advancing age (4, 5). exchange equal quantities of DNA. In an unequal T-SCE, one The G-rich nature of telomeric DNA renders it susceptible to sister telomere becomes longer at the expense of the other. Ini- G-quadruplex formation, oxidative damage, and alkylation by elec- tially, we suggested that in telomerase-deficient backgrounds trophiles (6). Telomeres are also unique in their chromatin com- unequal T-SCE might confer a proliferative growth advantage to position, being bound by the shelterin complex and associated pro- a subset of cells that stochastically acquired longer telomeres, en- teins, their T-loop structure, and their nucleosomal organization (7, 8). Telomeres protect genetic information by providing a buffer against the end-replication problem, but their terminal position at Author contributions: R.T.H., L.J.N., E.H.G., and S.M.B. designed research; R.T.H., K.B.B., L.J.N., the ends of linear DNA poses a special challenge to their own re- and S.M.B. performed research; R.T.H., K.B.B., L.J.N., E.H.G., and S.M.B. analyzed data; and plication (9). Telomerase is a ribonucleoprotein complex consisting R.T.H., K.B.B., L.J.N., E.H.G., and S.M.B. wrote the paper. minimally of RNA template and catalytic reverse transcriptase The authors declare no conflict of interest. subunits responsible for de novo synthesis of telomeric repeats (10). *This Direct Submission article had a prearranged editor. In the absence of telomerase, telomeres shorten with each cell di- 1To whom correspondence should be addressed. E-mail: [email protected]. vision (11). Critically short telomeres fail to form proper protective This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. end structures, which are sensed as DSBs and trigger a permanent 1073/pnas.1006338107/-/DCSupplemental. 15768–15773 | PNAS | September 7, 2010 | vol. 107 | no. 36 www.pnas.org/cgi/doi/10.1073/pnas.1006338107 Downloaded by guest on October 1, 2021 abling their escape from cellular senescence (16). Conversely, those also displayed normal background T-SCE frequencies (0.29 T-SCE cells inheriting shorter telomeres from an unequal exchange would per chromosome) (Fig. 1). be at risk for triggering senescence prematurely. Using a theoretical To evaluate the consequence of WRN and BLM deficiency on model and Monte Carlo simulations (23), we investigated the pos- T-SCE frequencies in the absence of telomerase, we used an RNA sibility that ALT arises from elevated T-SCE levels and concluded interference siRNA strategy to knock down their expression in it was unlikely unless there was an additional mechanism, such as normal human primary fibroblasts (telomerase-negative). The has been proposed by others (14, 24), through which longer telo- WRN-depleted cells displayed no significant increase in G-SCE meres segregate preferentially into the same daughter cell. This frequencies vs. the mock control (0.12 vs. 0.11) (Fig. 2), although study (23) strongly suggested that in ALT cells, T-SCE coexists with a statistically significant (P < 0.05) increase in T-SCE was ob- served (0.81 vs. 0.25) (Fig. 2). These results are consistent with our an as yet unknown telomere-lengthening mechanism. − − − − SCE is a relatively common event in normal human cells that previous study using cells derived from Wrn / ,Terc / double- occurs along the entire length of a chromosome. Although rec- knockout mice (19). ognized for over 50 y (25), their biological significance remains neither fully understood nor appreciated. SCE are induced by BLM Suppresses SCE Recombination Globally. In contrast to WS lym- DNA-damaging agents that block DNA replication (26), and can phoblasts, human